The Role of Actin and Tubulin on the Formation of Membrane Blebs in Rat Hepatocytes

• Main Author: 江明才
• Other Authors: 李宗貴
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/83138658423105657217

碩士 === 中山醫學院 === 毒理學研究所 === 86 === 　　Normal metabolismn and drug metabolism in cellular microenvirement can lead to the production of reactive oxygen species. Once these reactive these reactive oxygen species cannot be eliminated, cells will be damaged and leads to membrane morphological changes, such as the bleb formation. The purpose of this study was to examine the cellular surface blebs of oxidative damage. The important is cytoskeletal proteins-actin and tubulin acts in cellular surface blebs. Besides, intracellular important antioxidants: ascorbic acid, the study was compare the protective effects of ascorbic acid on the membrane morphological changes. The study was two section. 　　In the first section of the study, the examine the role of membraneassociated cytoskeletal proteins during the process of hepatocytes blebbing. After developing 48 hours, hepatocytes enters 2.0 mM t-butyl hydroperxide (t-BHP) or 2.0 mM diamide (thiol oxidant) to induced cell blebbing. The samples collected at 0, 15, 30, 60 minutes to analyze cell about membrane protein thiol deletion; the leakage of lactate dehydrodenase; the change of glutathione; the lipid peroxidation; the variation of actin and tubulin, and use Confocal fluorescent microscope to observe the change of actin and tubulin in rat hepatocytes. The result indicates the t-BHP or diamide both cause the mass loss about the membrane protein thiol in hepatocytes, the fast oxidization about glutathione in hepatocytes, the leakage of lactate dehydrodenase, but the level of t-BHP is larger than of diamide. In the aspect of lipid peroxidation, only the t-BHP produces apparent change. As to actin and tubulin, they both find fall-off and present time-dependency effects. The result in Confocal fluorescent microscope also show these two cytoskeletal proteins are related to cell blebbing, but they don''t play the same role during the porcess. Tubulin may be more important than actin. If the study predispose dithiothreitol (DTT, thiol reductant) before adding t-BHP or diamide, then the damage by the diamide is repressed, but just partly protection for the damage by t-BHP. The study reveals the change of cell type is related to thiol in cell, especially cytoskeletal proteins. 　　In the second part, the explore ascorbic acid if protecting cell type under disposing 2.0 mM t-BHP. The result finds that the damage of cell type and the condition of membrane protein thiol improve evidently under predisposing 500 μM ascorbic acid. But the aspect of lipid peroxidation is not repressed, it shows ascorbic acid can decrese the blebbing derived by t-BHP, and the mechanism is related to ascorbic acid which protects membrane protein, especially cytoskeletal proteins, from oxidization. 　　In a word, we think: 1)Under oxidization stress, the oxidization of membrane protein thiol may play more inportant role in cell blebbing. 2)Actin and tubulinare related to the maintenance of cell type, and tubulin may be more important than actin. 3)ascorbic acid can repress cell type to change under oxidization stress.